EN ISO 21479:2020

Soil quality - Determination of the effects of pollutants on soil flora - Leaf fatty acid composition of plants to assess soil quality (ISO 21479:2019)

This document describes a method to compare the quality of soils by determining the fatty acid composition of the leaves of plant species grown in these soils.
This method does not make it possible to determine an optimal value of the Omega-3 index and, therefore, cannot be used to determine the intrinsic quality of a soil from a specific area (regarded as homogeneous). The method can only be used to compare the quality of soils between various areas.
This method is applicable to:
—          soils from contaminated sites;
—          amended soils;
—          soils after remediation;
?      soil with waste products (e.g. slurry, manure, sludge or composts).
Alternatively, the quality of soils can be assessed by determining the Omega-3 index of Lactuca sativa seedlings grown in these soils under controlled conditions (i.e. phytotronic chamber) and by comparing these values to those obtained from control soils (see Annex B).

Bodenbeschaffenheit - Bestimmung der Wirkungen von Schadstoffen auf die Bodenflora - Fettsäurezusammensetzung in Blättern zur Beurteilung der Bodenbeschaffenheit (ISO 21479:2019)

Gegenstand dieses Dokuments ist ein Verfahren zum Vergleichen der Beschaffenheit von Böden durch Bestimmen der Fettsäurezusammensetzung in den Blättern von Pflanzen, die auf diesen Böden wachsen.
Dieses Verfahren ermöglicht keine Bestimmung des optimalen Werts des Omega 3 Index. Daher kann es nicht dazu verwendet werden, die intrinsische Qualität des Bodens auf einer bestimmten (als homogen angesehenen) Fläche zu bestimmen. Das Verfahren kann nur dazu verwendet werden, die Beschaffenheit von Böden auf verschiedenen Flächen miteinander zu vergleichen.
Das Verfahren ist anwendbar auf:
   Böden von kontaminierten Standorten;
   verbesserte Böden;
   Böden nach Altlastensanierungen;
   Böden, auf die Abfallprodukte (z. B. Flüssigmist, Gülle, Schlamm oder Kompost) aufgebracht wurden.
Alternativ kann die Beschaffenheit von Böden beurteilt werden, indem der Omega 3 Index von Lactuca sativa Keimlingen bestimmt wird, die in diesen Böden unter kontrollierten Bedingungen (d. h. in einer Wachstumskammer) gezogen werden, und diese Werte mit den bei Kontrollböden erhaltenen Werten verglichen werden (siehe Anhang B).

Qualité du sol - Détermination des effets des polluants sur la flore du sol - Composition en acides gras foliaires des plantes utilisées pour évaluer la qualité du sol (ISO 21479:2019)

Le présent document décrit une méthode visant à comparer la qualité des sols en déterminant la composition en acides gras des feuilles d'espèces végétales poussant sur ces sols.
Cette méthode ne permet pas de déterminer une valeur optimale de l'indice Oméga-3 et ne peut donc pas être utilisée pour déterminer la qualité intrinsèque d'un sol d'une zone spécifique (considérée homogène). La méthode peut être utilisée uniquement pour comparer la qualité des sols entre plusieurs zones.
Cette méthode est applicable à:
—          des sols provenant de sites contaminés;
—          des sols amendés;
—          des sols après remédiation;
—          des sols contenant des produits résiduaires (par exemple lisier, fumier, boues ou composts).
La qualité des sols peut aussi être évaluée en déterminant l'indice Oméga-3 de plantules de Lactuca sativa poussant dans ces sols dans des conditions contrôlées (c'est-à-dire, enceinte phytotronique) et en comparant ces valeurs avec celles obtenues à partir de sols témoins (voir l'Annexe B).

Kakovost tal - Določanje učinkov onesnaževal na floro tal - Sestava maščobnih kislin v listih rastlin za oceno kakovosti tal (ISO 21479:2019)

General Information

• Status: Published
• Publication Date: 05-May-2020
• Withdrawal Date: 29-Nov-2020
• ICS: 13.080.30 - Biological properties of soils
• Technical Committee: CEN/TC 444 - Environmental characterization
• Drafting Committee: CEN/TC 444 - Environmental characterization
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 06-May-2020
• Due Date: 07-Mar-2022
• Completion Date: 06-May-2020
• Ref Project: SIST EN ISO 21479:2020 - Soil quality - Determination of the effects of pollutants on soil flora - Leaf fatty acid composition of plants to assess soil quality (ISO 21479:2019)

Overview

EN ISO 21479:2020 (ISO 21479:2019) specifies a bioanalytical method to assess soil quality by measuring the leaf fatty acid composition of plants grown in soils of interest. The method uses the Omega‑3 index (leaf fatty acid profiles, expressed as FAMEs) as a sensitive bio‑indicator of pollutant effects on soil flora. It is intended for comparative assessments between areas (e.g., contaminated vs. reference sites) and cannot establish an absolute or intrinsic soil quality threshold.

Key topics and technical requirements

• Scope and limitation
  • Designed to compare soil quality across areas; not to define an optimal Omega‑3 index for a homogeneous soil.
  • Applicable to contaminated soils, amended soils, soils after remediation, and soils with waste products (slurry, manure, sludge, composts).
• Sampling and sample handling
  • Guidance for leaf tissue sampling strategies and contamination control.
  • Only small amounts of fresh leaf tissue are required (typically 20–50 mg), minimizing plant damage.
  • Plant tissues can be stored in methanol for several days before analysis.
• Analytical workflow
  • Extraction and conversion to fatty acid methyl esters (FAMEs) and instrumental analysis (chromatography) of FAME composition.
  • Annexes provide details on apparatus, reagents, analysis procedures, and example chromatograms.
• Quality and reproducibility
  • Ring test results (Annex A) address method repeatability and reproducibility.
  • Annex B describes an ex situ option: measuring the Omega‑3 index of Lactuca sativa seedlings grown under controlled (phytotronic) conditions and compared with controls.
• Safety
  • Warning about handling contaminated soils - potential toxic or infectious hazards; follow occupational health precautions.

Applications

• Detecting sub‑lethal effects of metals, herbicides and organic contaminants on plant physiology.
• Monitoring the effectiveness of soil remediation or amendments by comparing pre/post-treatment soil-induced changes in plant fatty acids.
• Screening contaminated or waste‑amended soils in environmental site assessments and long‑term monitoring programs.
• Complementary tool to chemical analyses and biometric endpoints (germination, biomass), offering greater sensitivity in some cases.

Who should use this standard

• Environmental laboratories and analytical chemists performing soil bioassays.
• Soil scientists, ecotoxicologists, remediation consultants and regulatory agencies assessing contaminated land.
• Researchers studying plant responses to soil pollutants and practitioners running site monitoring programs.

Related standards

• Part of the ISO family for soil quality (developed by ISO/TC 190). Use alongside chemical and physical soil testing standards and other biological characterization standards for comprehensive site assessment.

Keywords: soil quality, ISO 21479, leaf fatty acid composition, Omega‑3 index, contaminated sites, remediation, FAMEs, Lactuca sativa.

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2020
Kakovost tal - Določanje učinkov onesnaževal na floro tal - Sestava maščobnih
kislin v listih rastlin za oceno kakovosti tal (ISO 21479:2019)
Soil quality - Determination of the effects of pollutants on soil flora - Leaf fatty acid
composition of plants to assess soil quality (ISO 21479:2019)
Bodenbeschaffenheit - Bestimmung der Wirkungen von Schadstoffen auf die Bodenflora
- Fettsäurezusammensetzung in Blättern zur Beurteilung der Bodenbeschaffenheit (ISO
21479:2019)
Qualité du sol - Détermination des effets des polluants sur la flore du sol - Composition
en acides gras foliaires des plantes utilisées pour évaluer la qualité du sol (ISO
21479:2019)
Ta slovenski standard je istoveten z: EN ISO 21479:2020
ICS:
13.080.30 Biološke lastnosti tal Biological properties of soils
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 21479
EUROPEAN STANDARD
NORME EUROPÉENNE
May 2020
EUROPÄISCHE NORM
ICS 13.080.30
English Version
Soil quality - Determination of the effects of pollutants on
soil flora - Leaf fatty acid composition of plants to assess
soil quality (ISO 21479:2019)
Qualité du sol - Détermination des effets des polluants Bodenbeschaffenheit - Bestimmung der Wirkungen
sur la flore du sol - Composition en acides gras foliaires von Schadstoffen auf die Bodenflora -
des plantes utilisées pour évaluer la qualité du sol (ISO Zusammensetzung von Fettsäuren in Blättern zur
21479:2019) Beurteilung der Bodenbeschaffenheit (ISO
21479:2019)
This European Standard was approved by CEN on 13 April 2020.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 21479:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 21479:2019 has been prepared by Technical Committee ISO/TC 190 "Soil Quality” of the
International Organization for Standardization (ISO) and has been taken over as EN ISO 21479:2020 by
Technical Committee CEN/TC 444 “Environmental characterization of solid matrices” the secretariat of
which is held by NEN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by November 2020, and conflicting national standards
shall be withdrawn at the latest by November 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 21479:2019 has been approved by CEN as EN ISO 21479:2020 without any modification.

INTERNATIONAL ISO
STANDARD 21479
First edition
2019-06
Soil quality — Determination of the
effects of pollutants on soil flora —
Leaf fatty acid composition of plants
used to assess soil quality
Qualité du sol — Détermination des effets des polluants sur la flore du
sol — Composition en acides gras foliaires des plantes utilisées pour
évaluer la qualité du sol
Reference number
ISO 21479:2019(E)
©
ISO 2019
ISO 21479:2019(E)
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 21479:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and abbreviated terms . 1
3.1 Terms and definitions . 1
3.2 Abbreviated terms . 2
4 Principle . 2
5 Apparatus and reagents . 2
5.1 Apparatus . 2
5.2 Reagents. 3
6 Sampling strategies . 3
7 Sampling of leaf tissues . 3
8 Obtaining, extraction and analyses of FAMES . 4
8.1 Contamination control . 4
8.2 Obtaining and extraction of FAMES from plant leaves . 4
8.3 Analysis of FAMES . 4
9 Test report . 6
9.1 A reference to this document, i.e. ISO 21479 . 6
9.2 Description of the site and areas analysed . 6
9.3 Leaf sampling . 6
9.4 Fatty acid composition . 6
9.5 Conclusion . 6
Annex A (informative) Results of the ring test . 7
Annex B (informative) Assessment of soil quality by determining the Omega-3 index of
Lactuca sativa seedlings grown ex situ under controlled conditions .14
Annex C (informative) Plant species previously successfully used to assess soils of
contaminated sites (organic and/or metals) .16
Annex D (informative) Variation of the Omega-3 index as function of harvest time, plant
size and leaf development .17
Annex E (informative) Effect of the quantity of foliar tissues on the FAMES composition .19
Annex F (informative) Example of chromatogram obtained after the FAMES analysis of
foliar tissues .20
Annex G (informative) Recommended mathematical method to rate soils of areas when
some sampled plant species are not found in all areas .21
Bibliography .23
ISO 21479:2019(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 190, Soil quality, Subcommittee SC 4,
Biological characterization.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
iv © ISO 2019 – All rights reserved

ISO 21479:2019(E)
Introduction
Among the more than 150 ISO standards on soil quality that have been developed, less than 40 address
living organisms, and among them only five address higher plants. This is despite the importance of
monitoring the adverse effects of soil quality on living organisms.
[1]
One of these five standards addresses genotoxicity , and four of them address emergence and/or
[2-5]
growth inhibition . It therefore appears that these International Standards are focused either on a
very specific effect (genotoxicity), or on effects great enough to induce developmental (and, therefore,
visible) phenotypes (emergence or growth inhibition of young seedlings) in soils sampled in the field.
Hence, more sensitive/earlier bio-indicators of the adverse effects of pollutants on plants, such as the
“Omega-3 index”, are needed.
The assessment of soil contaminant effects by the Omega-3 index is based on the leaf fatty acid
composition of angiosperm species grown in sites of concern. The use of the Omega-3 index has proven
to be appropriate for highlighting the presence of metallic and organic contaminants (herbicides, etc.)
in the soils. With this aim, physical and chemical properties (pH, N/P/K content) of soils should also be
[12]
determined because plant fatty acid composition may vary as a function of nutrient content and pH
may influence chemical compound bioavailability. It should be noted that this bio-indicator has proved
to be more sensitive (i.e. responding to lower doses of contaminants) than the biometric parameters of
[6][14]
rate of germination and biomass . Hence, this makes it possible to gain evidence of adverse effects
of soils on plants that could not be highlighted by the rate of germination or biomass. Additionally, for in
situ assessment purposes, it can be difficult to observe evident effects on the rate of germination and/
or biomass of plants.
It should be noted that from a practical point of view, especially with plant species harvested in the
field, and in comparison with other bio-indicators, the Omega-3 index presents several advantages.
— For fatty acid analysis, only 20 mg to 50 mg of fresh leaf tissues per sample are needed. Hence, this
is not destructive for plants, and there is not a problem with getting enough tissues of one species
from a given area.
— Samples of plant tissues can be stored in methanol for several days at room temperature prior to
analyses.
— It is not necessary to find a particular species at a site, and that a priori any species (often chosen
among the most representative) can be sampled (Clause 6).
The results of a ring test performed by six individual laboratories to assess the reproducibility and the
repeatability of the method are shown in Annex A. The results obtained by the same investigator with
the same sample and the same measuring instrument over a short period of time are shown in Annex B.
INTERNATIONAL STANDARD ISO 21479:2019(E)
Soil quality — Determination of the effects of pollutants on
soil flora — Leaf fatty acid composition of plants used to
assess soil quality
WARNING — Contaminated soils can contain unknown mixtures of toxic, mutagenic, or
otherwise harmful chemicals or infectious micro-organisms. Occupational health risks can
arise from dust or evaporated chemicals. Furthermore, plants might take up chemicals from the
soil and safety measures should also be considered when handling the test plants.
1 Scope
This document describes a method to compare the quality of soils by determining the fatty acid
composition of the leaves of plant species grown in these soils.
This method does not make it possible to determine an optimal value of the Omega-3 index and,
therefore, cannot be used to determine the intrinsic quality of a soil from a specific area (regarded as
homogeneous). The method can only be used to compare the quality of soils between various areas.
This method is applicable to:
— soils from contaminated sites;
— amended soils;
— soils after remediation;
— soil with waste products (e.g. slurry, manure, sludge or composts).
Alternatively, the quality of soils can be assessed by determining the Omega-3 index of Lactuca sativa
seedlings grown in these soils under controlled conditions (i.e. phytotronic chamber) and by comparing
these values to those obtained from control soils (see Annex B).
2 Normative references
There are no normative references in this document.
3 Terms, definitions and abbreviated terms
3.1 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1.1
Omega-3 index
% C18:3/(%C18:0 + % C18:1 + % C18:2)
Note 1 to entry: The Omega-3 index has no unit.
ISO 21479:2019(E)
3.2 Abbreviated terms
For the purposes of this document, the following abbreviated term applies.
FAME(s) Fatty Acid Methyl Ester(s); C16:0: palmitic acid methyl ester; C16:1: palmitoleic methyl
ester; C18:0: stearic acid methyl ester; C18:1: oleic acid methyl ester; C18:2: linoleic acid
methyl ester; C18:3: linolenic acid methyl ester.
4 Principle
The method is used to assess the quality of soils by determining the fatty acid composition of the leaves
of angiosperm species (see Annex C and [9-14][18]) grown on these soils. After sampling leaf tissues,
their fatty acid composition is determined. For this, transesterification is carried out on the foliar
tissues and the fatty acid methyl esters obtained are analysed by gas chromatography. After analysis,
the % C18:3 / (% C18:0 + % C18:1 + % C18:2) ratio is calculated. The lower this ratio, the higher the
adverse effects on plants induced by soils is [6][9-14][18].
5 Apparatus and reagents
5.1 Apparatus
In addition to the standard laboratory equipment, the following apparatus are required.
5.1.1 Scissors to cut leaves.
5.1.2 Graduated glass pipette, to add sulfuric acid (H SO ) to methanol, pipettes to dispense the
2 4
mixture into glass culture tubes (1 ml/tube), and Pasteur pipettes for recovering hexane after extraction
of FAMEs.
5.1.3 Glass culture tubes (e.g. 1,3 × 10 cm) with polytetrafluoroethylene seal screw caps. These culture
tubes were numbered on adhesive tape (and not directly on the glass, to prevent any risk of erasing).
Tubes were checked to ensure they were not chipped (in order to guarantee their leak-tight seal).
5.1.4 System (e.g. heating block) for heating the tubes to 80 °C.
5.1.5 Benchtop centrifuge for centrifuging the tubes to 200 g to 300 g and separating the aqueous
phase from hexane.
5.1.6 Gas chromatograph vials with inserts and screws caps with a polytetrafluoroethylene septum.
5.1.7 Gas chromatograph equipped with a Flame Ionisation Detector (FID) and a capillary column for
separating and quantifying methyl esters of fatty acids with 12 carbon atoms to 22 carbon atoms, and for
each aliphatic chain length to separate the saturated, mono-, di- and tri-unsaturated esters.
Note 1 Most of the time, the studies that led to the preparation of this document were carried out using a gas
chromatograph (Hewlett Packard 5890 series II or Hewlett Packard 7890A) on a Carbowax 1,2 micron, 0,53 mm
diameter, 15 m long capillary column (Altech, Deerfield, IL., USA) or on a DB-WAX 1 micron, 0,53 mm diameter,
1)
15 m long capillary column (Agilent, Santa Clara, CA., USA), helium being the carrier gas .
1) This information is given for the convenience of users of this standard and does not constitute an endorsement
by ISO of these products.
2 © ISO 2019 – All rights reserved

ISO 21479:2019(E)
5.2 Reagents
5.2.1 Methanol (99 %) and sulfuric acid (H SO ), components of the transesterification solution.
2 4
5.2.2 Distilled water and hexane (99 %) for extracting the FAMEs.
6 Sampling strategies
Because plant fatty acid composition can vary as a function of climatic conditions, the compared areas
should share the same climatic conditions (humidity, temperature, sunlight). In addition, because the
Omega-3 index is an early indicator, its measurement is not relevant when a strong visual phenotype
(highly reduced biomass, high leaf chlorosis, etc.) is detected for plants having grown in one area, and
not detected in another area.
Depending on the aim of the study, one or several angiosperm species can be sampled from each area
of interest. For most of the studies, even if only one species can be used for the assessment of a given
site (a metallurgic landfill soil for example), it is recommended to use several species (if possible three
to eight). By using only one species, it is possible to serendipitously sample a highly resistant (or
sensitive) species. In addition, the larger the number of species sampled, the more representative the
results will be of a “soil quality” for the overall phytocoenosis. Hence, in this case, the various areas
of the site are first prospected, and species to sample are chosen among the most representative
examples, common to all areas to the extent possible. One leaf (or a piece of a leaf when whole leaves are
too large to be entirely immerged in 1 ml of methanol/H SO , see 8.2) from four to eight individuals per
2 4
species should be sampled per area. Some plant species previously successfully used to assess the soils
of contaminated sites (by organic compounds and/or metals) are indicated in Annex C.
When it is not possible to sample the same species in all the areas, it remains possible to determine
the Omega-3 index but, in this case: (i) all the species sampled in a given area should be present and
sampled on at least one other area and (ii) all pairs of areas should share at least one species to be
sampled.
Note that for the assessment of agricultural practices, the only plant species to sample is usually the
only one of interest, namely the cultivated crop. When only one species is sampled, the leaf (or a piece of
leaf) of 6 to 12 individuals per area is harvested.
7 Sampling of leaf tissues
The following recommendations should be followed to sample leaf tissues suitable for subsequent
analysis:
— as the transesterification response involves obtaining fatty acid methyl esters from biological
samples, and the presence of water leads to hydrolysis of the esters formed, the presence of external
water on the biological samples must be avoided. Hence, if leaves are wet, before sampling, it is
necessary to remove water from their surface by the use of an absorbent paper;
— do not sample leaves under hydric (drought) or biotic (pathogens) stress. Only green leaves should
be harvested;
— harvest leaves on plants of similar size. Consequently, harvest of leaves from small plants in one
area and leaves from tall plants in another should not be undertaken to measure the Omega-3 index
(see Annex D and [12]);
— as a precautionary measure, we recommend harvesting only mature leaves and to disregard
developing ones (see Annex D);
— when only a part of the leaves from a given species is sampled, harvest the same part of the leaves
(the distal part for example) for all individuals;
ISO 21479:2019(E)
— as a precautionary measure, it is recommended to harvest all the plants within 2 h to 3 h (see Annex D).
8 Obtaining, extraction and analyses of FAMES
8.1 Contamination control
To prevent contamination, it is necessary to avoid any contact between the solutions with plastic, parafilm
or glue, etc. To ensure the absence of contaminations (e.g. protocol errors, contaminated solutions, etc.),
a test should be performed before each series of analyses by following the same protocol described in 8.2
and 8.3, but without biological tissues in the culture tubes. After GC analyses, with the exception of the
peak corresponding to hexane, the profile of the gas chromatogram should not display peaks.
Avoid any contact of the solutions with plastic: this recommendation does not apply to the pipette tips
used for collecting the solution of methanol/H SO (40/1) or hexane.
2 4
8.2 Obtaining and extraction of FAMES from plant leaves
Introduce the foliar tissues (approximately 1 cm × 1 cm) into the culture tubes (see 5.1.3) containing
1 ml of a solution of methanol/H SO (40/1). Seal the tubes using a screw cap equipped with a
2 4
polytetrafluoroethylene seal. Heat them for 1 h at 80 °C. With the methanol boiling at 72 °C at a pressure
of 1 atmosphere, it is mandatory to avoid any evaporation so as to cause saturation vapour pressure in
the tubes. It is, therefore, important that they are perfectly plugged. It is also necessary to visually
check (every 5 min for 20 min, then every 10 min) that the solution of methanol/H SO does not boil
2 4
for the duration of the heating. If during the heating the contents of the tube boil, lower the tube into
the ice to cool it then completely unscrew and retighten the cap. Readjust the volume, if necessary, to
1 ml by adding methanol. If the contents are still boiling afterwards, take another tube and another cap
and transfer into it the contents of the defective tube. The fatty acid composition of tissues in the tubes
where the solution of methanol/H SO has (almost) totally evaporated during the heating should not be
2 4
analysed.
After 1 h of heating at 80 °C, cool the tubes (e.g. put them on ice). First add 750 µl of 99 % hexane,
then 1,5 ml of H O. Shake vigorously by hand for 20 sec. The use of a vortex should be avoided.
Centrifuge the tubes at 200 g to 300 g for 5 min to 10 min to obtain two phases. Using a Pasteur pipette,
transfer 200 µl to 400 µl of hexane (upper phase) into a CG vial equipped with an insert. Close the vial
using a screw-opening cap equipped with a silicone septum. Collecting the lower phase should absolutely
be avoided because the water irreversibly damages the column used for the gas chromatography.
Note that following this protocol, the leaf fatty acid composition does not depend on the amount of
foliar tissues put in the tube (see Annex E).
8.3 Analysis of FAMES
Carry out the gas chromatography analysis with a capillary column for separating and quantifying fatty
acid methyl esters with 14 carbon atoms to 22 carbon atoms, and for each aliphatic chain length for
separating the saturated, mono-, di- and tri-unsaturated esters. The FAMEs are identified by comparing
retention times with standard of C16:0; C16:1; C16:3 C18:0; C18:1; C18:2 and C18:3 methyl esters.
After the gas chromatography analysis (see Annex F), consider the surface of the peaks of the
chromatograph corresponding to C16:0, C16:1, C16:3 (when present), C18:0, C18:1, C18:2 and C18:3.
Express the results as a percentage for each FAME F . The percentage is calculated by dividing the
i
4 © ISO 2019 – All rights reserved

ISO 21479:2019(E)
surface S of the peak for the FAME F by the sum of the surfaces of the peaks corresponding to C16:0,
i i
C16:1, C16:3 (when present), C18:0, C18:1, C18:2 and C18:3, i.e.
% F = 100 × S /(S + S + S + S + S + S + S) (1)
i i C16:0 C16:1 C16:3 C18:0 C18:1 C18:2 C18:3
The results of the analyses for which a contamination is suspected should be discarded. For example,
when the fatty acid composition deviates too far from the standard fatty acid composition for green
leaf tissues of angiosperms (see Annexes B, E, F and [6][9-14][18]).
— %C18:0, %C16:1 and %C18:1 should each be lower than 10 %;
— C16:0 and C18:2 each between 5 % and 30 %;
— C16:3 between 0 % and 30 %; and
— C18:3 higher than 40 % of the sum (%C16:0 +%C16:1 + %C16:3 + %C18:0 + %C18:1 + %C18:2 +
%C18:3).
Similarly do not consider results when (an) additional peak(s) that do(es) not correspond to C16:0,
C16:3, C18:0, C16:1, C18:1, C18:2 or C18:3 appear(s) in significant proportions, e.g. peak(s) displaying a
surface S higher than 0,15 × (S + S + S + S + S + S + S ).
C16:0 C16:1 C16:3 C18:0 C18:1 C18:2 C18:3
One or several plant species can be sampled per area. When only one plant species is analysed or when
all plant species are found in all sampling areas, standard statistical procedures generally are sufficient
for analysis of results. The parametric analyses (e.g. t-test) for such data assume that the data are
normally distributed, that the treatments are independent and that the variance is homogenous among
the various treatments. These assumptions should be tested. If the data satisfy these assumptions,
results analysis may proceed. Otherwise, nonparametric tests should be used. Solely in cases of
statistically significant differences between areas, a rating is attributed to the soil of each area.
When only one species is analysed, and if significant differences between the means of the Omega-3
index by area are observed, the rating of an area should be defined as the mean of the Omega-3 index
on this area divided by the highest mean of the Omega-3 index measured on the whole site, all areas
included.
The Omega-3 index values may depend upon the plant species analysed. Therefore, when several plant
species are analysed together, standardised values of the Omega-3 index should be used. The X /X
p max
ratio is therefore calculated, where X represents the Omega-3 index measured for the individual plant
p
p and X represents the highest Omega-3 index obtained for the individuals of the same species at a
max
given site, all areas included.
When several plant species are analysed and all plant species were found in all sampling areas, and if
significant differences are observed between the means of the X /X ratio by area (all individuals
p max
of all species included), the rating of an area is defined as the mean of the X /X on this area (all
p max
individuals of all species included) divided by the highest mean of X /X measured at a given site, all
p max
areas included.
Note that it remains possible to rate and to rank areas when not all plant species under study were
found in all sampling areas when (i) all species sampled on a given area were present and sampled on
at least one other area, and (ii) all pairs of areas shared at least one of the same sampled species. The

method to calculate ratings of the areas in this case is fully described in [10] and explained in Annex G.
It should be noted that ratings are relative: for a given site, the area with the “best soil quality” is given
[6][9-14][18]
a rating of 1, other areas are given a lower rating. In view of our previous experience , we
usually distinguish the different adverse effects of soil on plants as follows: little or no effect (rating
≥0,93), medium (0,93> rating ≥0,85), high (0,85> rating ≥0,7) and very high adverse effect (0,7> rating),
relative to soil of the area with a reference rating of 1.
ISO 21479:2019(E)
9 Test report
The test report should include the following information:
9.1 A reference to this document, i.e. ISO 21479
9.2 Description of the site and areas analysed
When available, a detailed map of the given site is joined to the test report. Mention area by area whether
few or many plant species were present, and the most abundant ones. Note that detailed information on
physical and chemical properties is helpful for the interpretation of the results (see Introduction).
9.3 Leaf sampling
— Area by area, the plant species sampled (when possible: Linnaean classification, variety, source), the
number of individuals from each of these species and when appropriate, missing individual(s);
— The part of the leaves sampled (distal, proximal), and the leaves sampled (the mature leave, the
oldest leaf, etc.); and
— Any specific particularity of the status of the sampled individual (developmental stage, age, size,
etc.) and when appropriate description of visual damage (photographs are recommended).
9.4 Fatty acid composition
— When appropriate, missing data (broken tubes, contaminated sample, etc.);
— The fatty acid composition of each sample and the value of the % C18:3 / (% C18:0 + % C18:1 +
% C18:2) ratio, its mean and standard deviation; and
— When several species are analysed, the standardised % C18:3 / (% C18:0 + % C18:1 + % C18:2)
ratio, its mean and standard deviation species by species.
9.5 Conclusion
Conclusions on the relative soil quality of the various areas are drawn following analysis of results as
described in 8.2 and 8.3.
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ISO 21479:2019(E)
Annex A
(informative)
Results of the ring test
A.1 General
Six laboratories (noted A, B, C, D, E and F) from three European countries (France, Portugal and the
United Kingdom) were involved in the ring test.
The sampling campaign was carried out on May 31, 2017 for all participants. The site under study
(27 m × 19 m) was located at Léognan, near Bordeaux (France). It was divided into several areas (of
3 m × 6 m) with each area separated by 1 m. Leaf tissues were harvested from plants growing on three
different areas: Area 2, Area 7 and Area 20. These areas displayed various metal content (see Table A.1)
because Areas 2 and 7 had received municipal digested and dehydrated sewage sludge between 1974
and 1993 (10 t per hectare every year for Area 2 and 100 t per hectare every second year for Area 7),
whereas Area 20 did not receive any sewage sludge.
Table A.1 — Total content of metal trace elements (mg/kg)
Total Metal Content (mg/kg)
Area
Cd Cr Cu Ni Pb Zn
2 6,01 21,9 85,8 23,6 179 977
7 10,3 33,8 135 39 307 1790
20 (Control) 0,62 10,9 34,2 4,03 48,6 55,5
Every laboratory, A, B, C, D, E and F, synthesised, extracted and analysed FAMEs in the Laboratoire
de Biogenèse Membranaire (LBM, Bordeaux). The results obtained by the various investigators in
Bordeaux are indicated with letters A1, B1, C1, D1, E1 and F1, respectively.
Moreover, laboratories A, B, C, and D additionally synthesised, extracted and analysed FAMEs in their
own laboratories; the corresponding results are indicated with A2, B2, C2 and D2, respectively.
Only three plant species were found in all the areas and analysed: Plantago lanceolata, Hypochaeris
radicata and Medicago sativa. For each series (A1 to F1 and A2 to D2), laboratories independently
harvested five pieces of leaf from five individuals for each species (Figures A.1 and A.2).
After fatty acid analyses, the mean of the standardised Omega-3 index values was calculated area by
area, all species included.
ISO 21479:2019(E)
a) With Plantago lanceolata
b) With Hypochaeris radicata
c) With Medicago sativa
Key
P indicates the parts of the leaf sampled by investigators performing analyses solely at the LBM
Figure A.1 — Plant species harvested and protocol of leaf sampling for the analyses only
performed at the LBM (one sample)
8 © ISO 2019 – All rights reserved

ISO 21479:2019(E)
a) With Plantago lanceolata
b) With Hypochaeris radicata
c) With Medicago sativa
Key
P1 and P2 represent the parts of the leaf sampled by investigators who carried out analyses both at the LBM (with
parts P1) and at their own laboratory (with parts P2)
Figure A.2 — Plant species harvested and protocol of leaf sampling for the analyses performed
at the LBM and in the other laboratories (two samples)
A.2 Results obtained in Bordeaux by six laboratories
Figure A.3 shows that in Areas 20 and 7, all laboratories found similar mean of standardised Omega-3
index values of the: 0,781 ± 0,016 (Kruskal-Wallis test: p = 0,883) in Area 20, and 0,786 ± 0,037
(Kruskal-Wallis test: p = 0,269) on Area 7. Green dots represent outliers.
ISO 21479:2019(E)
Area 20
Area 7
Key
X laboratory involved in the test
Y normalized Omega-3 index
Figure A.3 — Standardised Omega-3 Index obtained for Areas 20 and 7 for all laboratories
in the LBM
By contrast in Area 2, a higher Omega-3 index, slightly but significantly higher than others, was
determined for laboratory B (see Figure A.4).
10 © ISO 2019 – All rights reserved

ISO 21479:2019(E)
Key
X laboratory involved in the test
Y normalized Omega-3 index
Kruskal-Wallis test:
a
p < 0,02
b
p < 0,01
c
p < 0,002
Figure A.4 — Standardised Omega-3 Index obtained for Area 2 for all laboratories in the LBM
To summarise, except for one investigator in one area (slightly higher Omega-3 index values than others),
on each area, all laboratories obtained the same standardised Omega-3 index values. Because values
were very close, it remains possible that among the 18 areas analysed (three areas x six laboratories),
the highest and lowest values of the standardised Omega-3 index were uniformly distributed among
the 17 areas, whereas one area randomly displayed a slightly greater highest value.
A.3 Results obtained by the same investigator in different laboratories:
As mentioned, laboratories A, B, C and D synthesised, extracted and analysed FAMEs from these samples
in their own laboratories. Unfortunately, the laboratory D did not use a suitable GC column; therefore,
it was not possible to take these results into consideration. In addition, laboratory C analysed the fatty
acid composition by GC-MS and not by GC-FID in its laboratory. We discuss below the results obtained
by GC-MS by laboratory C, but they cannot be directly compared to the results obtained in Bordeaux by
GC-FID analyses.
Table A.2 shows that in each area, each laboratory found the lowest (orange boxes) and highest (green
boxes) values associated with the same individual (all species included) in Bordeaux and in their
laboratories, with the exception of one value (blue box). In addition, both of the laboratories, A and B,
found similar standardised Omega-3 index values in Bordeaux (LBM) and in their own laboratories: all
species and all areas included, the ratios A1/A2 and B1/B2 were 1,008 ± 0,088 (n = 45) and 1,025 ± 0,079
(n = 45) for the samples analysed by the laboratories A and B respectively. This evidence shows the
low variability of the measurements when the same sample (i.e. the same leaves) was analysed in two
different locations (by the same investigator).
Table A.2 — Comparisons of standardised Omega-3 Index values measured on the same leaf at
LBM and the other laboratories and p-value of Kruskal-Wallis tests
Area 20 7 2
Laboratory A B A B A B
Sample N° A1 A2 B1 B2 A1 A2 B1 B2 A1 A2 B1 B2
1 0,58 0,51 0,88 0,82 0,84 0,82 0,99 0,800 0,78 0,71 0,99 1,00
2 0,73 0,62 0,92 0,88 1,00 1,00 0,82 0,77 0,83 0,89 0,97 0,89
ISO 21479:2019(E)
Table A.2 (continued)
Area 20 7 2
Laboratory A B A B A B
Sample N° A1 A2 B1 B2 A1 A2 B1 B2 A1 A2 B1 B2
3 0,79 0,73 0,79 0,73 0,92 0,89 0,87 0,91 0,82 0,78 0,96 0,94
4 0,91 0,81 0,69 0,69 0,81 0,80 1,00 1,00 0,88 0,92 0,86 0,74
5 0,70 0,64 0,93 0,94 0,86 0,90 0,82 0,72 0,75 0,79 0,96 0,93
6 1,00 1,00 0,73 0,78 0,85 0,92 0,85 0,87 0,67 0,80 0,68 0,62
7 0,87 0,91 0,70 0,86 0,85 0,98 0,77 0,82 0,58 0,56 0,84 0,84
8 0,76 0,81 0,82 0,77 0,61 0,61 0,98 0,95 0,73 0,76 0,98 1,00
9 0,74 0,78 0,82 0,91 0,59 0,60 0,96 0,92 0,77 0,79 0,88 0,92
10 0,80 0,82 0,76 0,80 0,63 0,66 1,00 1,00 0,67 0,72 0,97 0,98
11 0,83 0,74 0,89 0,74 0,73 0,70 0,78 0,77 1,00 1,00 1,00 0,92
12 0,79 0,77 0,82 0,81 0,81 0,83 0,65 0,69 0,89 0,91 0,86 0,81
13 0,70 0,81 0,82 0,73 0,74 0,73 0,69 0,70 0,85 0,80 0,97 0,96
14 0,74 0,71 0,71 0,85 0,90 0,58 0,57 0,89 0,93 0,78 0,67
15 0,78 0,86 0,68 0,67 0,88 0,82 0,73 0,79 0,64 0,67 0,99 1,00
Mean 0,780 0,773 0,798 0,789 0,798 0,811 0,832 0,819 0,783 0,802 0,913 0,882
SD 0,099 0,123 0,083 0,081 0,116 0,124 0,131 0,124 0,113 0,115 0,094 0,121
C 0,126 0,159 0,104 0,103 0,146 0,152 0,157 0,151 0,144 0,143 0,103 0,137
V
p-value 0,884 0,724 0,836 0,724 0,561 0,494
A.4 Outcome
Overall, results obtained following GC-FID analyses show that laboratories did not find any difference
between areas, regardless of where the analyses were performed (except for laboratory B who found a
slight, yet significant, difference between Area 2 and Area 20 for analyses conducted in Bordeaux, but
not for those conducted in their laboratory). Results obtained by GC-FID and GC-MS cannot be directly
compa
...